A cysteine-rich domain of the "mannose" receptor mediates GalNAc-4-SO4 binding.

A critical element of lutropin bioactivity in vivo is its rapid removal from the blood by a receptor, located in hepatic endothelial cells, that recognizes the terminal sulfated carbohydrate structure SO4-4-GalNAcbeta1,4GlcNAcbeta1,2Manalpha (S4GGnM). We have previously shown that the macrophage mannose (Man)-receptor cDNA directs the synthesis of a protein that binds oligosaccharides with either terminal S4GGnM or terminal Man, at independent sites. We now show that the cysteine-rich (Cys-Rich) domain at the N terminus of the Man/S4GGnM receptor accounts for binding of oligosaccharides with terminal GalNAc-4-SO4, whereas calcium-dependent carbohydrate recognition domains (CRDs) account for binding of ligands containing terminal Man. The Cys-Rich domain is thus a previously unrecognized carbohydrate binding motif. Cys-Rich domains have been described on the three other members of the endocytic C-type lectin family of receptors. The structural relationship of these receptors to the Man/S4GGnM receptor raises the possibility that their Cys-Rich domains also bind carbohydrate moieties and contribute to their function.

The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcbeta1,4GlcNAcbeta or Man at independent sites.

Lutropin (LH) and other glycoproteins bearing oligosaccharides with the terminal sequence SO4-4-GalNAcbeta1,4GlcNAcbeta1,4Man- (S4GGnM) are rapidly removed from the circulation by an S4GGnM-specific receptor (S4GGnM-R) expressed at the surface of hepatic endothelial cells. The S4GGnM-R isolated from rat liver is closely related to the macrophage mannose-specific receptor (Man-R) isolated from rat lung both antigenically and structurally. The S4GGnM-R and Man-R isolated from these tissues nonetheless differ in their ability to bind ligands bearing terminal GalNAc-4-SO4 or Man. In this paper, we have explored the structural relationship between the Man-R and the S4GGnM-R by examining the properties of the recombinant Man-R in the form of a transmembrane protein and a soluble chimeric fusion protein in which the transmembrane and cytosolic domains have been replaced by the Fc region of human IgG1. Like the S4GGnM-R isolated from liver, the chimeric fusion protein is able to bind ligands terminating with GalNAc-4-SO4 and Man at independent sites. When expressed in CHO cells the recombinant Man-R is able to mediate the uptake of ligands bearing either terminal GalNAc-4-SO4 or terminal Man. We propose that the Man-R be renamed the Man/S4GGnM receptor on the basis of its multiple and independent specificities.

Isolation of the SO4-4-GalNAcbeta1,4GlcNAcbeta1,2Manalpha-specific receptor from rat liver.

Glycoproteins, such as the glycoprotein hormone lutropin (LH), bear oligosaccharides terminating with the sequence SO4-4GalNAcbeta1, 4GlcNAcbeta1,2Manalpha (S4GGnM) and are rapidly removed from the circulation by a receptor present in hepatic endothelial cells and Kupffer cells. Rapid removal from the circulation is essential for attaining maximal hormone activity in vivo. We have isolated a protein from rat liver which has the properties expected for the S4GGnM-specific receptor (S4GGnM-R). The S4GGnM-R is closely related to the macrophage mannose receptor (Man-R) both antigenically and structurally. At least 12 peptides prepared from the S4GGnM-R have amino acid sequences that are identical to those of the Man-R. Nonetheless, the ligand binding properties of the S4GGnM-R and the Man-R differ in a number of respects. The S4GGnM-R binds to immobilized LH but not to immobilized mannose, whereas the Man-R binds to immobilized mannose but not to immobilized LH. When analyzed using a binding assay that precipitates receptor ligand complexes with polyethylene glycol, the S4GGnM-R is able to bind S4GGnM-bovine serum albumin (S4GGnM-BSA) conjugates whereas the Man-R is not. In contrast both the S4GGnM-R and the Man-R are able to bind Man-BSA. Monosaccharides that inhibit binding of Man-BSA by the Man-R enhance binding by the S4GGnM-R. Oligosaccharides terminating with S4GGnM and those terminating with Man are bound at independent sites on the S4GGnM-R. The S4GGnM-R present in hepatic endothelial cells may account for clearance of glycoproteins bearing oligosaccharides terminating with S4GGnM and glycoproteins bearing oligosaccharides terminating with either mannose, fucose, or N-acetylglucosamine.

Equine lutropin and chorionic gonadotropin bear oligosaccharides terminating with SO4-4-GalNAc and Sia alpha 2,3Gal, respectively.

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are heterodimeric glycoprotein hormones which are synthesized in the placenta and pituitary, respectively. The beta subunits of eCG and eLH, like their alpha subunits, arise from a single gene and have identical amino acid sequences. In contrast, the beta subunits of CG and LH in primates arise from different genes and differ in sequence. We have examined the structures of the Asn-linked oligosaccharides on eCG and eLH. eCG bears di- and tri-branched Asn-linked oligosaccharides terminating with Sia alpha 2,3 or 6Gal beta 1,4GlcNAc. In contrast, > 72% of the Asn-linked oligosaccharides on eLH have 1 or 2 branches terminating with the sequence SO4-4-GalNAc beta 1,4GlcNAc. The nonsulfated oligosaccharides on eLH are neutral (6% of the total) or have branches terminating with sialic acid-Gal (22% of the total). Since the alpha and beta subunits of eCG and eLH both contain the tripeptide motif, Pro-Xaa-Arg/Lys, recognized by the glycoprotein hormone-specific GalNAc-transferase found in pituitary, expression of the GalNAc- and sulfotransferases must differ in the placenta and pituitary. eLH, but not eCG, is bound by the hepatic endothelial cell receptor specific for the sequence SO4-4-GalNAc beta 1,4GlcNAc. As a result, eLH is removed from the circulation 5.7-fold more rapidly than eCG and is selectively localized to the liver. Since the major structural difference between eCG and eLH is in the terminal glycosylation of their Asn-linked oligosaccharides and this has a major impact on circulatory half-life, it is likely that the difference in circulatory half-life defines the functional difference between eCG and eLH.

The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha.

Tissue factor pathway inhibitor (TFPI) produced by endothelial cells contains sulfated Asn-linked oligosaccharides. We have determined that greater than 70% of the oligosaccharides on recombinant TFPI expressed in 293 cells terminate with the sequence SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha. Oligosaccharides terminating with this sequence have previously been described on lutropin, thyrotropin, and pro-opiomelanocortin: glycoproteins synthesized in the anterior pituitary. A GalNAc-transferase that recognizes the tripeptide motif Pro-Xaa-Arg/Lys 6-9 residues N-terminal to Asn glycosylation sites accounts for the specific addition of GalNAc to the oligosaccharide acceptor on these glycoproteins, whereas a GalNAc beta 1,4GlcNAc beta 1, 2Man alpha-4-sulfotransferase accounts for the addition of sulfate. The sulfated oligosaccharides present on these hormones are responsible for their rapid clearance from plasma by a receptor in hepatic reticuloendothelial cells. GalNAc- and sulfotransferase activities with the same properties as those expressed in the pituitary are detected at high levels in 293 cells and at lower levels in endothelial cells. Chinese hamster ovary (CHO) cells do not contain detectable levels of either transferase and rTFPI expressed in CHO cells does not contain sulfated Asn-linked oligosaccharides. TFPI contains the sequence Pro-Phe-Lys, 9 residues N-terminal to the glycosylation site at position 228; this tripeptide may act as the recognition sequence for the GalNAc-transferase. rTFPI produced by 293 cells, but not that produced by CHO cells, is bound by the receptor on hepatic reticuloendothelial cells suggesting the sulfated structures play a role in the biologic behavior of TFPI.

A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin.

We have identified a receptor in hepatic endothelial and Kupffer cells that binds oligosaccharides terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2-Man alpha (S4GGnM). This receptor can account for the rapid removal of the glycoprotein hormone lutropin, which bears unique Asn-linked oligosaccharides terminating in S4GGnM, from the circulation. Hepatic endothelial cells express 579,000 S4GGnM receptors at their surface and bind lutropin with an apparent Kd of 1.63 x 10(-7) M. Bound ligand is rapidly internalized. Binding does not require divalent cations, is reversed by incubation at pH 5.0 or below, and is inhibited by fucoidin but not by hyaluronate, heparin, chondroitin sulfate, or dextran sulfate. We propose that the S4GGnM-specific receptor represents a major mechanism for clearance of certain sulfated glycoproteins from the blood, including members of the glycoprotein hormone family.

Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes.

Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.

Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor.

The ligand-binding activity of the galactose/N-acetylgalactosamine-specific receptor (Gal/GalNAc receptor) present on the surface of hepatocytes can be modulated under a number of conditions in the intact cell. The carboxylic acid ionophores monensin and nigericin inhibit endocytosis by the Gal/GalNAc receptor in a concentration-dependent manner. Monensin at a concentration of 100 microM reduces the number of binding sites for asialo-orosomucoid and a tri-branched glycopeptide (F2) 5-10-fold; however, the number of Gal/GalNAc receptor subunits detected at the cell surface by a competitive radioimmunoassay and by immunoprecipitation of surface labeled receptor is not significantly altered. Replacement of NaCl in the medium with either N-methylglucamine or sorbitol to isotonicity also inhibits binding and endocytosis. The monensin, nigericin, N-methylglucamine, and sorbitol treatments have in common the ability to alkalinize the cytosol of the hepatocyte. None of these agents has any effect on binding by the isolated Gal/GalNAc receptor nor is the intracellular pH shift of such a magnitude that it would alter binding by the isolated Gal/GalNAc receptor. This has led us to conclude that the ligand-binding properties of the Gal/GalNAc receptor at the cell surface can be modulated in a transmembrane fashion by events other than those involving pH or Ca2+ regulation at the ligand-binding site itself. Such transmembrane modulation of ligand binding by the Gal/GalNAc receptor may provide a rapid and efficient mechanism for mediating ligand release and immediate return of the receptor to the cell surface.

Recycling of the hepatocyte asialoglycoprotein receptor does not require delivery of ligand to lysosomes.

The hepatocyte asialoglycoprotein receptor is able to mediate the internalization of ligand in buffer devoid of Na+ but containing 0.15 M K+. Under these conditions, degradation of internalized ligand does not occur due to an inability to deliver the ligand to lysosomes. Instead, the ligand becomes localized in a vesicle with the same density as plasma membrane on Percoll gradients. This vesicle may be the functional equivalent of the uncoated vesicles observed by electron microscopy. Internalization of more than 20 glycoprotein molecules/high affinity surface receptor was observed under these conditions, indicating that delivery of ligand to lysosomes is not necessary for receptor reutilization.

Photoactivatable glycopeptide reagents for site-specific labeling of lectins.

Photoactivatable, iodinated glycopeptides bearing oligosaccharides of defined structure have been synthesized for use as lectin binding site-specific agents. Two such glycopeptides have been examined utilizing concanavalin A, RCAI (Ricinus communis agglutinin), RCAII (R. communis toxin), and the Gal/GalNAc-specific lectin from human and rat hepatocytes. Covalent incorporation upon photoactivation only occurs with a glycopeptide which is specifically bound by the lectin and is inhibited only by appropriate haptenic monosaccharides. The efficiency of covalent coupling is on the order of 2%. Half-maximal covalent incorporation occurs at a concentration of photoactivatable glycopeptide which corresponds to the previously determined association constant for lectin binding in each case. Covalent incorporation of glycopeptide is accompanied by a decrease in the relative mobility of the lectin upon sodium dodecyl sulfate polyacrylamide gel electrophoresis equivalent to that expected for addition of an oligosaccharide moiety. The high degree of specificity and the relatively high affinity of these photoactivatable glycopeptides makes them promising agents for the examination of lectins such as the Gal/GalNAc-specific receptor present on mammalian hepatocytes.

Galactose and N-acetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes.

We have examined the kinetics of binding and uptake of iodinated glycoproteins and glycopeptides bearing terminal Gal or GalNAc moieties in an isolated rat hepatocyte system. Asparagine-linked, triantennary complex oligosaccharides with three terminal Gal residues are endocytosed with the same kinetics as asialo-orosomucoid, whereas biantennary, complex oligosaccharides with one or two terminal Gal residues are not endocytosed Glycopeptides bearing as few as four O-glycosidically-linked Gal beta 1, 3GalNAc or GalNAc moieties are also rapidly endocytosed, while glycopeptides with one or two more closely spaced moieties are not endocytosed. All the endocytosable glycoproteins and glycopeptides have similar apparent dissociation constants and a similar number of binding sites on the surface of the intact hepatocyte. The ligand-binding properties of the receptor in the plasma membrane of intact cells differ from those of the solubilized receptor, suggesting that interaction with other as yet undefined cellular components confers the ability to discriminate among closely related oligosaccharide structures. This is consistent with a model in which only glycopeptides bearing terminal Gal or GalNAc residues that fall within a restricted spatial relationship can induce a conformational alteration in the receptor which is required for uptake to occur. The endocytosis of a number of glycoproteins such as human asialo-ceruloplasmin can be accounted for by the presence of a single, complex oligosaccharide with the appropriate structure.

Structure of the complex oligosaccharides of fetuin.

The complete structure of the complex oligosaccharides of fetuin has been established. The three fractions of complex oligosaccharide which were isolated by ion exchange chromatography following pronase digestion (F-I, F-II, and F-III) had identical molar ratios of sialic acid (Sia), galactose, mannose, and N-acetylglucosamine of 3:3:3:5. A combination of methylation analyses, Smith periodate degradations, and endoglycosidase and exoglycosidase digestions were utilized to establish the structure which is proposed to be: (see article of journal). Features of this structure not previously established include the presence of 2 residues of alpha2,3- and 1 residue of alpha2,6-linked sialic acid and their location relative to the mannose branch points. Also unusual is the presence of an alpha-linked branch mannose with substituents at positions 2 and 4 which is in turn linked to position 6 of the beta-linked, branch mannose. These features result in unexpected resistance to specific exoglycosidases.